Quantitative evaluation of Mycobacterium abscessus clinical isolate virulence using a silkworm infection model

Mycobacterium abscessus causes chronic skin infections, lung diseases, and systemic or disseminated infections. Here we investigated whether the virulence of M. abscessus clinical isolates could be evaluated by calculating the median lethal dose (LD50) in a silkworm infection model. M. abscessus subsp. abscessus cells were injected into the silkworm hemolymph. When reared at 37˚C, the silkworms died within 2 days post-infection with M. abscessus subsp. abscessus. Viable cell numbers of M. abscessus increased in the hemolymph of silkworms injected with M. abscessus. Silkworms were not killed by injections with heat-killed M. abscessus cells. The administration of clarithromycin, an antibacterial drug used to treat the infection in humans, prolonged the survival time of silkworms injected with M. abscessus. The LD50 values of 7 clinical isolates in the silkworm infection model were differed by up to 9-fold. The Mb-17 isolate, which was identified as a virulent strain in the silkworm infection model, induced more detachment of human THP-1-derived macrophages during infection than the Mb-10 isolate. These findings suggest that the silkworm M. abscessus infection model can be used to quantitatively evaluate the virulence of M. abscessus clinical isolates in a short time period.


Introduction
The Mycobacterium abscessus complex (MABC) is a group of rapidly growing non-tuberculous mycobacteria that includes 3 subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii [1][2][3][4]. The MABC causes chronic skin infections, lung diseases, and systemic or disseminated infections in immunocompromised patients [5][6][7][8]. The virulence of MABC has evolved through a stepwise adaptation to the host and soil environments [5,9], and could thus vary between clinical isolates. Several mouse MABC infection models have been used to evaluate the efficacies of antibacterial drugs [10,11]. Existing mouse models of MABC infection require several weeks to complete a single study, which is not convenient, especially for MABC virulence screening purposes. Thus, developing a model that permits a more rapid evaluation of MABC virulence is highly desirable. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Silkworms are invertebrate animals beneficial for use in experiments to reveal host-pathogen interactions [12][13][14]. A large number of silkworms can be reared in a small space compared with mammalian animals [15]. The 3Rs, replacement, refinement, and reduction, are important animal welfare principles for experiments using mammals [16]. Experiments using invertebrates are consistent with the concept of replacement. Because the silkworm is an invertebrate, fewer ethical issues are associated with the use of a large number of silkworms for experimentation compared with mammals. By exploiting this advantage of silkworms for infectious disease research, the median lethal dose (LD 50 ), which is the dose of a pathogen required to kill half of the animals in a group, can be determined to quantitatively compare the virulence of different strains [17,18]. Silkworm infection models are used as initial screening systems to identify virulence-related genes in pathogenic microorganisms [19][20][21][22]. Silkworm infection models are therefore useful for comparing the virulence of microorganisms [15]. A silkworm infection model was established to evaluate anti-mycobacterial compounds using a type strain [23]. Virulence among M. abscessus clinical isolates based on the LD 50 values, however, has not been evaluated in a silkworm M. abscessus infection model.
In the present study, we compared the virulence of M. abscessus subsp. abscessus clinical isolates by calculating the LD 50 values in a silkworm infection model. Among the 7 clinical isolates evaluated, the extent of the virulence varied up to 9-fold. Furthermore, using the silkworm infection model, the M. abscessus subsp. abscessus Mb-17 isolate was identified as a highly virulent strain that exhibits higher cytotoxic activity against human THP-1 macrophages compared with the Mb-10 isolate. These findings suggest that the silkworm infection model is a rapid evaluation system for quantitatively estimating the virulence of M. abscessus subsp. abscessus clinical isolates.

Bacterial strains and growth
The M. abscessus subsp. abscessus ATCC19977 strain and 7 clinical isolates (Mb-7, Mb-10, Mb-14, Mb-16, Mb-17, Mb-18, and Mb-22) were used in this study. The clinical isolates were obtained from sputum samples of patients infected with M. abscessus subsp. abscessus at the Keio University School of Medicine. This study was approved by the medical research ethics committee of the National Institute of Infectious Diseases (#1046) and by the Keio University School of Medicine Ethics Committee (#2008-0131-9 sai). Bacterial species were identified with a DDH Mycobacteria Kit (Kyokuto Pharmaceutical Industrial Co., Tokyo, Japan) [24] and multiplex polymerase chain reaction [25]. The M. abscessus subsp. abscessus strains were grown on a Middlebrook 7H10 agar plate at 37˚C. A single colony was then inoculated into 5 ml of Middlebrook 7H9 broth and incubated at 37˚C for 3 days.

Infection experiments using silkworms
The silkworm infection experiments were performed as previously described [26]. Fifth instar larvae were reared on an artificial diet (Silkmate 2S, Ehime-Sanshu Co., Ltd., Ehime, Japan) for 24 h. M. abscessus subsp. abscessus cells grown in Middlebrook 7H9 broth were collected by centrifugation and suspended in sterile saline. A 50-μl of sample solutions was administered to the silkworm hemolymph by injecting the silkworm dorsally using a 1-ml tuberculin syringe (Terumo Medical Corporation, Tokyo, Japan). Silkworms were injected with the M. abscessus subsp. abscessus cells (1.4 x 10 7 cells per larva), and were incubated at 27˚C or 37˚C, and their survival was monitored.
The therapeutic activity of clarithromycin in silkworms was evaluated according to a previous study with slight modifications [26]. Either 50 μl of saline or 50 μl of an M. abscessus subsp. abscessus suspension was injected into the silkworm hemolymph. Clarithromycin (25 μg/g larva) was immediately injected into the silkworms. The silkworms were incubated at 37˚C, and their survival was monitored.

Viable cell counts
Silkworms were injected with an M. abscessus subsp. abscessus cell suspension (7 x 10 6 cells in 50 μl) and incubated at 37˚C. Hemolymph was harvested from the silkworm larvae through a cut on the first proleg at either 3 or 18 h post-infection [17]. The hemolymph was added to saline, and the solution was spread on a Middlebrook 7H10 agar plate. The agar plate was incubated at 37˚C for 3 days, and the colonies on the agar plate were counted.

LD 50 measurement
The LD 50 values were determined according to a previous study, with slight modifications [27,28]. M. abscessus subsp. abscessus cells grown in Middlebrook 7H9 broth were suspended in saline. Either a 2-or 4-fold dilution series of the bacterial suspension was prepared. The bacterial suspension (4 x 10 5 -1 x 10 8 cells/50 μl) was injected into the silkworm hemolymph, and the silkworms were incubated at 37˚C. The number of surviving silkworms was counted at 48 h after infection. The LD 50 values were determined from the data of 3 experiments using a simple logistic regression model in Prism 9 (GraphPad Software, LLC, San Diego, CA, USA, https://www.graphpad.com/scientific-software/prism/).

Cytotoxicity test using human THP-1-derived macrophages
To evaluate the cytotoxicities of M. abscessus subsp. abscessus clinical isolates against human THP-1-derived macrophages, a high-content imaging analysis was performed to count the number of cells attached to the polystyrene surface of a 96-well plate. Cell detachment from the well correlated with cell death [29]. Therefore, the cytotoxicities of the M. abscessus subsp. abscessus clinical isolates against THP-1-derived macrophages were determined by monitoring the cell detachment. Human THP-1 monocytes were cultured in RPMI1640 medium (Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific Inc., Waltham, MA, USA) and ampicillin (50 μg/mL) at 37˚C and 5% CO 2 . Cells (2 x 10 4 cells/well) were seeded into 96-well plates (Cell Carrier-96well Ultra microplate; PerkinElmer Inc., Waltham, MA, USA) and differentiated to macrophages with phorbol 12-myristate 13-acetate (10 ng/mL) for 72 h. The single monolayer THP-1 macrophages were infected by M. abscessus subsp. abscessus Mb-10 or Mb-17 isolates at a multiplicity of infection of 50, centrifuged at 1000 rpm for 5 min, and incubated for 4 h. To remove the extracellular bacteria, wells were washed twice with phosphate-buffered saline (PBS) and RPMI 1640 with amikacin (200 μg/mL) was added. After 2-h incubation, wells were washed twice with PBS and RPMI 1640 was added. The plates were incubated for 48 h at 37˚C and 5% CO 2 . After incubation, infected cells were fixed with 4%(v/v) paraformaldehyde for 10 min at room temperature and washed 3 times with ice-cold PBS before treating them with 0.1% Triton X-100 for 10 min, and then washing 3 times with PBS. The cells were stained with Hoechst 33258 (1:1000; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and HCS Cell Mask Deep Red (1:20000; Thermo Fisher Scientific Inc., MA, USA) for 25 min at room temperature and then washed 3 times with PBS. Stained cell images were obtained using a High-Content Imaging System Operetta CLS (PerkinElmer Inc.) with 40x Air/0.6 NA. The number of attached cells (cytoplasm with nuclei) was automatically calculated by Harmony software (PerkinElmer Inc.). Data are expressed as the mean ± the standard deviation (SD). Statistical analysis was performed with GraphPad Prism 9 (GraphPad Software).

Statistical analysis
The statistical significance of differences between viable cell counts of M. abscessus subsp. abscessus in silkworm groups were determined by the Student t-test. Statistically significant differences between survival curves in the silkworm infection experiments were evaluated using a log-rank test in GraphPad Prism 9 (GraphPad Software). The statistical significance between Mb-10 and MB-17 isolates in the infection experiment using human THP-1 macrophages was calculated using the Turkey test with one-way ANOVA. Values of P < 0.05 were considered significant.

Experimental conditions for the evaluation of M. abscessus subsp. abscessus virulence in silkworms
We first determined the experimental conditions for evaluating the virulence of M. abscessus subsp. abscessus in silkworms. In silkworm infection experiments, the rearing temperature is critical because it affects bacterial virulence and silkworm health [15]. The standard silkworm rearing temperature is 27˚C, and 37˚C corresponds to human body temperature. We previously reported that administration of Staphylococcus aureus (1 x 10 7 cells) to silkworms killed them in 3 days under rearing conditions of 27˚C [19]. Within 48 h of injection of M. abscessus subsp. abscessus ATCC19977 strain (1.4 x10 7 cells), silkworms died at 37˚C, but did not die at 27˚C (Fig 1A and 1B). The viable cell count of M. abscessus subsp. abscessus in the silkworm hemolymph at 18 h post-infection at 37˚C was higher than that at 27˚C (Fig 1C). The LD 50 for M. abscessus subsp. abscessus ATCC19977 was 1.1 x 10 7 cells under a 37˚C rearing condition (Fig 2). These results suggest that a 37˚C rearing condition is necessary for M. abscessus subsp. abscessus-induced silkworm death within 2 days.

Virulence effect of M. abscessus subsp. abscessus proliferation in silkworms
Because silkworm death caused by Porphyromonas gingivalis in a previous report did not require proliferation, we assumed that the silkworm death was caused by shock not induced by the bacterial infection [30]. We tested whether M. abscessus subsp. abscessus proliferates in the silkworm body, and if proliferation is necessary for virulence. The M. abscessus subsp. abscessus viable cell count increased in the silkworm hemolymph at 18 h post-infection (Fig 3). The injection of autoclaved M. abscessus subsp. abscessus cells did not kill silkworms (Fig 4A). Clarithromycin, a bacteriostatic antibiotic, is used to treat clinical M. abscessus subsp. abscessus infections in humans [8,31]. The administration of clarithromycin to silkworms infected with M. abscessus subsp. abscessus prolonged their survival time (Fig 4B). These results suggest that the virulence of M. abscessus subsp. abscessus against silkworms requires the growth of M. abscessus subsp. abscessus.

Evaluating the virulence of M. abscessus subsp. abscessus clinical isolates against silkworms
We next determined the LD 50 values of M. abscessus subsp. abscessus clinical isolates using the silkworm infection model to compare their virulence. Seven clinical isolates were obtained from sputum samples of patients infected with M. abscessus subsp. abscessus. Their LD 50 values ranged from 3.1 x 10 6 to 2.9 x 10 7 cells per larva, with the LD 50 value of the Mb-17 isolate being the lowest (Fig 5). The LD 50 value of the Mb-17 isolate was 9-fold lower than that of the Mb-10 isolate. The viable cell count of the Mb-17 isolate in the silkworm hemolymph at 18 h post-infection was higher than that of the Mb-10 isolate (Fig 6). These results suggest that the Mb-17 isolate has higher virulence against silkworms than the Mb-10 isolate.

Evaluating the cytotoxicities of M. abscessus subsp. abscessus clinical isolates against human THP-1-derived macrophages
Human THP-1-derived macrophages can adhere to the polystyrene surface of a 96-well plate. Chemical induced cell detachment from the well correlates with cell death [29]. We next determined the cytotoxicities of the Mb-10 and Mb-17 isolates against THP-1-derived macrophages by monitoring cell detachment. The number of macrophages attached to the well was decreased by infection with these strains at a multiplicity of infection of 50 (Fig 7). The Mb-17  isolate led to a decrease in the number of attached macrophages compared with the Mb-10 isolate (Fig 7). The result suggests that the Mb-17 isolate, which was identified as a highly virulent strain using the silkworm infection model, induces a greater detachment of THP-1-derived macrophages during infection than the Mb-10 isolate.

Discussion
In the present study, the virulence of M. abscessus subsp. abscessus clinical isolates was compared using a silkworm infection model. Among the 7 clinical isolates, the virulence, as determined by the LD 50 , varied up to 9-fold. These results indicate that the in vivo silkworm evaluation system is useful for revealing the virulence of M. abscessus subsp. abscessus clinical isolates with a short time period (2 days).
M. abscessus subsp. abscessus-infected silkworms incubated at 37˚C were more sensitive to infection than those reared at 27˚C. We assumed that this difference was due to both hightemperature stress in silkworms and the optimal growth temperature for M. abscessus subsp. abscessus. Hosoda et al. reported establishing a silkworm infection model for evaluating antimycobacterial compounds [23]. Here, we demonstrated that M. abscessus subsp. abscessus grows in the silkworm hemolymph and compared the virulence of several clinical isolates. Our findings are important toward validating the usefulness of the silkworm infection model for estimating the virulence of M. abscessus subsp. abscessus clinical isolates. The LT 50 value, which is the incubation time required to kill half of the silkworms in a group, differed slightly among time-course experiments. Therefore, a secondary evaluation to determine the LD 50 values based on multiple dose-dependent experiments is needed.
M. abscessus subsp. abscessus virulence may correlate with severity infection [5,9]. Therefore, understanding M. abscessus subsp. abscessus clinical isolate virulence is useful information for infection control. M. abscessus subsp. abscessus exhibited a different extent of virulence among clinical isolates in the silkworm model. We demonstrated that the silkworm infection model with M. abscessus subsp. abscessus is advantageous for quantitative determination of clinical isolate virulence by calculating the LD 50 values within 2 days. Moreover, the LD 50 values among the clinical isolates differed up to 9-fold. The Mb-17 isolate was the most virulent against silkworms among the M. abscessus subsp. abscessus clinical isolates used in this study. We hypothesized that the Mb-17 isolates harbor virulence-related genes that enhance the infection process. Moreover, the cytotoxicity of the Mb-17 isolate against human THP-1-derived macrophages was higher than that of the Mb-10 isolate. These results suggest that the Mb-17 isolate, which is highly virulent in silkworm infection model, is also highly cytotoxic to human macrophages. Future studies will include experiments aimed at revealing the relationship between the information on disease parameters in patients and the virulence against silkworms. The virulence genes of several pathogens have been identified by mutant screening using silkworm infection models from a mutant library [19][20][21][22]. The method for constructing M. abscessus subsp. abscessus gene-deletion mutants is well established [32][33][34]. Further studies are needed to determine the virulence factors harbored by the M. abscessus subsp. abscessus Mb-17 isolate that are responsible for its virulence in silkworms.
M. abscessus vertebrate infection models using a zebrafish, Danio rerio, mice, and a tadpole, Xenopus laevis, have been reported [33,35,36]. These infection models are used to evaluate anti-mycobacterial drugs and virulence within 15 days. The rearing temperature may be related to the experimental period. For example, infected zebrafishes were reared at 28-32˚C. In this study, infected silkworms died under a rearing condition at 37˚C within 2 days but not at 27˚C. Therefore, the silkworm has the benefit of being able to examine the infection experiments at 37˚C. Because these model animals including a zebrafish are vertebrates, however, more ethical problems are associated with their use with respect to animal welfare. The 3Rs, replacement, refinement, and reduction, are important principles for experiments using mammals [16]. Silkworms are invertebrate animals with several advantages as an alternative model animal for infection experiments requiring a high number of animals. Moreover, M. abscessus subsp. abscessus virulence factors relevant to human pathogenicity could be identified using this silkworm infection model.
Our study has some limitations. First, the silkworm infection model established in this study is not a respiratory infection model because the bacterial cells were injected into silkworm hemolymph. Therefore, the silkworm infection model deviates significantly from M. abscessus infection in humans. Second, it is unknown how the bacteria kill silkworms. Therefore, silkworm tissues targeted by the bacterial cells should be determined by histopathological studies.

Conclusion
We propose that the silkworm infection model with M. abscessus subsp. abscessus is an advantageous assay system for determining the virulence of M. abscessus subsp. abscessus clinical strains in a short time period. The silkworm infection model may contribute to revealing the molecular mechanisms of M. abscessus subsp. abscessus infections.
Supporting information S1 Dataset. Datasets included in this study. (XLSX)